Method and applications for efficient genetic suppressor elements

ABSTRACT

Methods for isolating and identifying genetic elements that are capable of inhibiting gene function are disclosed, as well as genetic elements isolated or identified according to the method of the invention and host cells modified by genetic modification using genetic suppressor elements according to the invention.

This application is a divisional of U.S. Ser. No. 08/039,385, filed Sep.7, 1993, now U.S. Pat. No. 5,811,234, issued Sep. 22, 1998, which isrelated as a U.S. National Phase application to PCT/US91/07492, havingan International Filing Date of Oct. 11, 1991, which application claimspriority under the terms of the Paris Convention and 35 U.S.C. §§120 and361 to U.S. Ser. No. 07/599,730, filed Oct. 19, 1990, now U.S. Pat. No.5,217,889, issued Jun. 8, 1993, the disclosures thereof being explicitlyincorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to means for suppressing specific gene function ineukaryotic or prokaryotic cells. More particularly the invention relatesto the use of expression of DNA sequences, known as genetic suppressorelements, for the purpose of suppressing specific gene function. Theinvention provides methods for obtaining such genetic suppressorelements, the genetic suppressor elements themselves, and methods forobtaining living cells which bear a gene suppression phenotype.

2. Summary of the Related Art

Functional inactivation of genes through the expression of specificgenetic elements comprising all or a part of the gene to be inactivatedis known in the art. At least four mechanisms exist by which expressionof such specific genetic elements can result in inactivation of theircorresponding gene. These are interference with protein function bypolypeptides comprising nonfunctional or partly nonfunctional analogs ofthe protein to be inhibited or a portion thereof, interference with mRNAtranslation by complementary anti-sense RNA or DNA, destruction of mRNAby anti-sense RNA coupled with ribozymes, and interference with mRNA byRNA sequences homologous to a portion of the mRNA representing animportant regulatory sequence.

Herskowitz, Nature 329: 219-222 (1987), reviews the inactivation ofgenes by interference at the protein level, which is achieved throughthe expression of specific genetic elements encoding a polypeptidecomprising both intact, functional domains of the wild type protein aswell as nonfunctional domains of the same wild type protein. Suchpeptides are known as dominant negative mutant proteins.

Friedman et al., Nature 335: 452-454 (1988), discloses the use ofdominant negative mutants derived from HSV-1 VP16 protein by 3′truncation of the VP16 coding sequence to produce cells resistant toherpes-virus infection. Baltimore, Nature 335: 395-396 (1988), suggeststhat the method might be applicable as a therapeutic means for treatmentof HIV-infected individuals.

Green et al., Cell 58: 215-223 (1989), discloses inhibition of geneexpression driven by an HIV LTR, through the use of dominant negativemutants derived from the HIV-1 Tat protein sequence, using chemicalpeptide synthesis.

Rimsky et al., Nature 341: 453-456 (1989), discloses inhibition ofHTLV-1 and HIV-1 gene expression in an artificial plasmid system, usingdominant negative mutants derived from the HTLV-1 Rex transactivatorprotein by oligonucleotide-mediated mutagenesis of the rex gene.

Trono et al., Cell 59: 113-120 (1989), demonstrates inhibition of HIV-1replication in a cell culture system, using dominant negative mutantsderived from the HIV-1 Gag protein by linker insertional and deletionalmutagenesis of the gag gene.

Ransone et al., Proc. Natl. Acad. Sci. USA 87: 3806-3810 (1990),discloses suppression of DNA binding by the cellular Fos-Jun proteincomplex and suppression of Jun-mediated transcriptional transactivation,using dominant negative mutants derived from Fax and Jun proteins byoligonucleotide-directed substitutional or deletional mutagenesis of thefos and jun genes.

Whitaker-Dowling et al., Virology 175: 358-364 (1990), discloses acold-adapted strain of influenza A virus which interferes withproduction of wild-type influenza A virus in mixed infections,apparently by a dominant negative mutant protein mechanism.

Lee et al., J. Bacteriol. 171: 3002-3007 (1989), discloses a geneticsystem for isolation of dominant negative mutations of the beta subunitof E. coli RNA polymerase obtained by hydroxylamine mutagenesis of therpoB gene.

Chejanovsky et al., J. Virol. 64: 1764-1770 (1990), discloses inhibitionof adeno-associated virus (AAV) replication by a dominant negativemutant protein derived from the AAV Rep protein byoligonucleotide-directed substitutional mutagenesis of the rep gene at aposition encoding an amino acid known to be critical to Rep proteinfunction.

Suppression of specific gene function by interference at the RNA level,using complementary RNA or DNA sequences, is also known in the art. vander Krol et al., BioTechniques 6: 958-976 (1988), reviews the use ofsuch “antisense” genes or nucleotide sequences in the inhibition of genefunction in insect, bird, mammalian, plant, protozoal, amphibian andbacterial cells.

Ch'ng et al., Proc. Natl. Acad. Sci. USA 86: 10006-10010 (1989)discloses that antisense RNA complementary to the 3′ coding andnon-coding sequences of the creatine kinase gene inhibited in vivotranslation of creatine kinase mRNA when expressed from a retrovirusvector, whereas all antisense RNAs complementary to creatine kinasemRNA, but without the last 17 codons or 3′ non-coding sequences, werenot inhibitory.

Daugherty et al., Gene Anal. Techn. 6: 1-16 (1989) discloses that, forantisense RNA suppression of beta galactosidase (β-gal) gene function inE. coli, best suppression is achieved using plasmids containing aribosome binding site and expressing short RNA sequences correspondingto the 5′ end of the β-gal gene.

Powell et al., Proc. Natl. Acad. Sci. USA 86: 6949-6952 (1989),discloses protection of transgenic plants from tobacco mosaic virus(TMV) when the plants expressed sequences complementary to replicasebinding sites, but not when they expressed sequences complementary onlyto TMV coat protein.

Sarver et al., Science 247: 1222-1225 (1990), discloses the use ofantisense RNA-ribozyme conjugates to degrade specific mRNA bycomplementary RNA binding followed by ribozyme cleavage of the boundmRNA.

Kerr et al., Eur. J. Biochem. 175: 65-73 (1988), reports that even fulllength antisense RNA is not necessarily sufficient to inhibit geneexpression.

Inhibition of gene function can also be accomplished by expressingsubregions of RNA which is homologous to, rather than complementary to,important regulatory sequences on the mRNA molecule, and which canlikely compete with the mRNA for binding regulatory elements importantto expression.

Bunnell et al., Somat. Cell Mol. Genet. 16: 151-162 (1990), disclosesinhibition of galactosyltransferase-associated (GTA) protein expressionby transcription of an RNA which is homologous to AU-rich elements(AREs) in the 3′ untranslated region of the gta gene, which are believedto be important regulatory sequences.

Although gene suppression is quite useful for scientific studies of genefunction and holds considerable promise for certain applications indisease therapy and genetic modification of plants and animals, currentmethods for identifying effective genetic suppressor elements (GSEs) aretime consuming and arduous. Interference by dominant negative mutantproteins, for example, either requires extensive knowledge about thefunctional domain structure of the protein so that reasonably promisingcandidate mutant proteins can be prepared, or necessitates individualpreparation and screening of numerous candidate mutant proteins.Antisense RNA and competitive homologous RNA similarly require extensiveindividual preparation and screening of candidate inhibitory sequences,absent considerable knowledge about important specific sequences withinthe RNA. There is, therefore, a need for generalized methods foridentifying and isolating GSEs which will allow simplified determinationof effective elements without undue experimentation or extensivestructure/function knowledge. An ideal method would allow simultaneousanalysis of multiple possible candidate GSEs, regardless of theirmechanism of action.

BRIEF SUMMARY OF THE INVENTION

The invention relates to the suppression of specific gene function ineukaryotic or prokaryotic cells. More particularly, the inventionrelates to nucleotide sequences which are capable of suppressing genefunction when expressed in a living cell. These nucleotide sequences areknown as genetic suppressor elements. Existing methods of suppressinggene function in living cells require considerable information about thestructure and function of the gene products, i.e., specific RNAsequences or specific protein domains. Alternatively, existing methodsof suppressing gene function can be applied in the absence of detailedstructure/function information, but at the expense of the considerabletime and effort required to produce many individual mutant proteins ormany complementary or homologous RNA or DNA sequences. In contrast, theinvention provides a general method for obtaining effective geneticsuppressor elements (GSEs) for cloned genes or viruses, withoutextensive structure/function information, and in a simple selection orscreening procedure.

The invention is made possible by two discoveries. First, the inventorshave discovered that small peptide fragments, corresponding to only aminute portion of a protein, can inhibit the function of that protein invivo, even without mutation of the fragments. Second, the inventors havedemonstrated that certain random small fragments of DNA, derived from aparticular gene or virus, are capable of inhibiting that particular geneor virus in vivo, when they are expressed in a living cell, and thatthese fragments can be isolated by functional selection for suppressionof the gene or virus.

In the method of the invention for obtaining GSEs, randomly fragmentedDNA, corresponding to DNA sequences from a gene or virus to beinactivated, is transferred into an expression library capable ofexpressing the random fragments of DNA in a living cell. Desired livingcells are then genetically modified by introducing into them the GSEexpression library by standard procedures, and cells containing GSEs areisolated or enriched for by selecting or screening for gene suppression.GSEs are then obtained from the living cells exhibiting the genesuppression phenotype.

GSEs obtained by the method of the invention may be used to geneticallymodify cells by introducing the GSE into the cell such that it can beexpressed and suppress gene function in the genetically modified cell.Alternatively, for some cell types it will be possible to obtaingenetically modified cells bearing a gene suppression phenotype as aresult of introduction of the GSE library, without ever having to firstisolate the GSE.

Genetically modified cells according to the invention can providebenefits, such as virus resistance, which can be commercially importantin biotechnology processes using living cells, as well as in food cropsderived from virus-resistant cells, or even in agriculturally importanttransgenic animals. In addition, improved agricultural plants andanimals can be produced from genetic modification by suppression ofgenes responsible for undesirable properties, e.g., cross-pollination ofinbred plants. Finally, genetic modification according to the inventionmay be useful for human therapeutic applications, such as antiviraltherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the distribution of GSEs in the lambda genome. Only thegenes whose sequences were found in GSEs are indicated in the geneticmap of lambda. Open bars indicate sense-oriented GSEs. Hatched barsindicate antisense-oriented GSES. The height of the bars corresponds tothe number of sequenced GSE clones for each class. The numbers on top ofthe bars indicate the extent of suppression of prophage induction by arepresentative clone of each class.

FIG. 2 shows the distribution of the oop/ori class of GSEs and thecorresponding lambda resistance phenotypes. Arrows indicate thedirection of transcription. The map position of the antisense ooptranscript is according to Krinke and Wulff, Genes Dev. 1: 1005 (1988).The four top clones were obtained by GSE selection. The two bottomclones were constructed by PCR synthesis using the correspondingprimers.

FIGS. 3 to 3C shows the nucleotide sequence of GSEs derived from humanTopoisomerase II, as described in Example 6: A is Seq ID No:1; B is SeqID No:2; C is Seq ID No:3; D is Seq ID No:4; E is Seq ID No:5; F is SeqID No:6; G is Seq ID No:7; H is Seq ID No:8; I is Seq ID No:9; J is SeqID No:10.

DETAILED DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Suppressing the function of specific genes by modifying cells to expressgene-specific inhibitory substances is an important approach to variousgoals in biotechnology and medicine. One of these goals is inhibition ofreplication of pathogenic viruses in genetically modified cells.

Other suppression targets include, for example, genes associated withtumorigenicity (oncogenes) as well as genes responsible for someundesired properties of agricultural plants or animals. Specificsuppression of a target gene requires expression of speciallyconstructed genetic elements that generally include modified DNAsequences derived from the target gene. In one of the currently usedapproaches to gene suppression, all or a portion of CDNA of the targetgene is inserted in a reverse orientation into an expression vectorcarrying a strong transcription promoter, so that antisense RNA istranscribed. Such antisense RNA can inhibit the function of the targetmRNA molecules. Certain genes may also be functionally suppressed byexpression of RNA sequences homologous to regulatory sequences in themRNA. In another, more recent approach, mRNA sequences in an antisenseorientation are combined with specific enzymatically active RNAsequences called ribozymes, which are capable of cleaving a target mRNAmolecule. Another way to suppress gene expression is to use a mutantform of the target protein that can act in a dominant negative fashionby interfering with the function of the wild-type (normal) form of thesame protein.

Although approaches to suppressing genes are thus known in the art,there are no general principles which provide guidance about how toderive DNA elements which can efficiently suppress gene function(genetic suppressor elements, or GSEs) without extensivestructure/function information about the RNA or protein, or withoutundue experimentation. The present invention provides a general methodfor obtaining GSES. The method of the invention requires only theavailability of genomic DNA, total cellular RNA, or of a cloned gene orDNA from a pathogenic virus or intracellularly parasitic microorganismtargeted for suppression and the knowledge of a selectable phenotypeassociated with inactivation of the target gene. This method does notdepend on any knowledge of the structure/function organization of theprotein encoded by the target gene or the genetic structure of thetarget virus or microorganism.

In a first aspect, the invention provides a convenient, general methodfor obtaining GSEs. In this method, purified DNA corresponding to thegene or genome to be suppressed is first randomly fragmented byenzymatic, chemical, or physical procedures. In a preferred embodiment,random fragments of DNA are produced by treating the DNA with anuclease, such as DNase I. The random DNA fragments are incorporated asinserts in a gene suppression element library, using an expressionvector which is capable of expressing the inserted fragments in the celltype in which gene suppression is desired. For general principles ofDNase I partial digestion and library construction see MolecularCloning, A Laboratory Manual, Sambrook et al., Eds., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989). In certain embodiments theinserted fragment may be expressed as part of a fusion protein. In otherembodiments the inserted fragment alone may be expressed. In anotherembodiment, ribozyme-encoding sequences may be inserted directlyadjacent to the insert to allow for selection of most efficientribozyme-antisense clones. In still other embodiments the genesuppression element library may be further modified by randommutagenesis procedures known in the art. The inserted fragments may beexpressed from either a constitutive or an inducible promoter.

The GSE library is next used to genetically modify living cells of thetype in which gene suppression is desired, by introducing the libraryinto the cells by procedures well known in the art, e.g., bacterial oryeast transformation, or transfection of plant or mammalian cells. See,e.g., Keown et al., Methods Enzymol. 185: 527-536 (1990). Of particularinterest in mammalian cells is the use of retroviral vectors such asLNCX (Miller and Rosman, Biotechniqes 7:980-986 (1989)); lambda ZD35,Murphy and Efstatiadis, Proc. Natl. Acad. Sci. USA 84: 8277-8281; orderivatives of convenient existing vectors, such as lambda Zap II®(Stratagene, LaJolla, Calif.) that have had inserted sequences thatallow retrovirus gene expression. The genetically modified cellscontaining effective GSEs can be screened for or selected in a varietyof ways. For example, when the suppression is directed against acytolytic virus, cells containing effective GSEs may be selected on thebasis of cell survival upon virus infection and development ofcytopathic effect. In another embodiment, suppression is directedagainst a non-cytolytic virus or against a gene encoding a cell surfaceantigen. In this embodiment, selection is against the presence of theviral or cell surface antigens. This is accomplished by reacting thegenetically modified cells with specific primary antibodies against theviral or cell surface antigens. “Unsuppressed” cells may then beeliminated by the addition of complement, or may be separated from“suppressed” cells by addition of fluorescent secondary antibody againstthe primary antibody, followed by fluorescence-activated cell sorting.For a general description of immunological selection and screeningtechniques see Davis et al., Microbiology, Harper and Row, Philadelphia,Pa. (1980). In another embodiment, suppression is directed against genesthat must be expressed in order for cells to grow under specificprocedures. In this embodiment, cells containing effective GSEs can beselected by “suicide selection” procedures that select for cells whichcannot grow in the selective medium. See Patterson et al., MethodsEnzymol. 151: 121 (1982).

In yet another embodiment, suppression is directed againstgrowth-suppressing genes, such as tumor suppressors. In this embodiment,cells containing effective GSEs may be screened on the basis ofmorphological transformation of cell colonies.

The GSE is finally obtained from the selected cells by procedures knownin the art. In one embodiment, the GSE is isolated by use of thepolymerase chain reaction with DNA obtained from the selected cells andwith primers homologous to sites on the vector flanking the insert. Inanother embodiment, the GSE expression library may be prepared inshuttle vectors, allowing efficient recovery of shuttle vectorscontaining GSEs (See, e.g., Groger et al., Gene 81: 285-294 (1989); Rioet al., Science 227: 23-28 (1985) for examples of shuttle vectors). Ofcourse, in bacteria simple plasmid isolation procedures can be employeddirectly on the bacterial clone expressing the genetically suppressedphenotype. Finally, GSEs can be isolated by standard cloning techniqueswell known in the art using vector specific probes although this mightbe more laborious than other embodiments herein described.

In a second aspect, the invention provides GSEs which are most likelymore effective than existing GSEs, since GSEs obtained according to themethod of the invention may be selected from a very large number ofpossible DNA sequences, whereas existing GSEs have been the result oftrial and error analysis of only a few designs. GSEs obtained accordingto the methods of the invention may operate according to principlesdifferent from those behind existing gene suppression methods, since itis the gene suppression phenotype, and not the mechanism, which isselected. GSEs obtained according to the methods of the invention areuseful for the genetic modification of living cells for scientificstudies, for biotechnology processes, for agricultural purposes and forhuman and animal therapeutic purposes. In addition, oligonucleotide oroligopeptide GSEs can be readily prepared which correspond to thenucleotide or amino acid sequence of the GSE obtained according to themethod of the invention. These oligonucleotides, which may be standardoligonucleotides, standard oligodeoxynucleotides or chemically modifiedderivatives of oligonucleotides or oligodeoxynucleotides, will becapable of inhibiting specific gene function, by virtue of homology tothe identified GSE. Such oligonucleotide inhibitors will be particularlyuseful for pharmaceutical purposes.

In a third aspect, the invention provides genetically modified livingcells that contain effective GSEs, whereby in such cells particulargenes are suppressed by the expression of the GSEs. In a preferredembodiment, such genetically modified cells are produced by introducinginto the cell, by standard procedures, an expression vector containing aspecific GSE obtained by the method of the invention and capable ofexpressing the GSE in the cell. In another embodiment the geneticallymodified cell is obtained directly from selection of cells into whichthe GSE library has been introduced, without any previous isolation ofthe GSE contained in the genetically modified cell.

In a fourth aspect, the invention provides a convenient method fordiscovering GSE, associated with a particular phenotype, rather thanwith a particular known gene. In this aspect, the method provides GSEscorresponding to recessive genes that, when inactivated, confer aselectable or screenable phenotype upon a cell having such inactivegenes. This method uses a random fragment expression system aspreviously described. However, the starting material is different. GSEsin this case are isolated from random fragment expression librariesprepared from either genomic DNA or total cellular cDNA. When used toobtain bacterial or lower eukaryotic GSEs, genomic DNA is preferred, forreasons of convenience. In contrast, cDNA is preferred for GSEs fromhigher eukaryotes, due to its lower complexity.

In a fifth aspect, the invention provides synthetic peptides andoligonucleotides that are capable of inhibiting the function ofparticular gene products. Synthetic peptides according to the inventionhave amino acid sequences that correspond to amino acid sequencesencoded by GSEs according to the invention. Synthetic oligonucleotidesaccording to the invention have nucleotide sequences corresponding tothe nucleotide sequences of GSEs according to the invention. Once a GSEis discovered and sequenced, and its orientation is determined, it isstraightforward to prepare an oligonucleotide corresponding to thesequence of the GSE (for antisense-oriented GSES) or to prepare apeptide corresponding to an amino acid sequence encoded by the GSE (forsense-oriented GSEs). In certain embodiments, such synthetic peptides oroligonucleotides may have the complete sequence encoded by the GSE orpresent in the GSE, respectively. In certain other embodiments, thepeptide or oligonucleotide may have only a portion of the GSE-encoded orGSE sequence. In such latter embodiments, undue experimentation isavoided by the observation that many independent GSE clonescorresponding to a particular gene will have the same 5′ or 3′ terminus,but generally not both. This suggests that many GSEs have one criticalendpoint, from which a simple walking experiment will determine theminimum size of peptide or oligonucleotide necessary to inhibit genefunction. For peptides, functional domains as small as 6-8 amino acidshave been identified for immunoglobulin binding regions. For antisenseoligonucleotides, inhibition of gene function can be mediated byoligonucleotides having sufficient length to hybridize to theircorresponding mRNA under physiological conditions. Generally,oligonucleotides having about 12 or more bases will fit thisdescription. Those skilled in the art will recognize that peptidemimetics and modified oligonucleotides are equivalent to the peptidesand oligonucleotides according to the invention, since both can beprepared according to standard procedures once the sequence necessaryfor inhibition is known.

The following examples are provided as means for illustration and arenot limiting in nature.

EXAMPLE 1 Suppression of Gene Function by Expression of a DNA SequenceEncoding a Small Polypeptide

P-glycoprotein, the product of the human mdr1 gene, is a multidrugtransporter that renders mammalian cells resistant to various lipophilicdrugs by pumping these drugs out of cells. See Chen et al., Cell 47: 381(1986). A short segment of mdr1 cDNA, corresponding to exon 7 of themdr1 gene and encoding a 57 amino-acid long peptide, was inserted bystandard procedures into an expression vector (pneoMLV), containing aG418-resistance gene, neo, as a selectable marker. One of the constructs(construct 1) was made in such a way that the mdr1-derived sequence waspreceded by the translation initiation codon at the 5′ end. At the 3′end, this sequence was adjoined to an open reading frame present in thevector sequence, so that the mdr1-derived sequence formed the N-terminalportion of the resulting fusion peptide. In another construct (construct2), the mdr1-derived sequence was preceded by the initiation codon andfollowed by a stop codon, giving rise to an entirely mdr1-derived 58amino acid protein (including the initiating methionine). Constructs 1and 2, as well as a control pSV2neo plasmid, were transfected into humanKB-8-5 cells, which display a moderate amount of multidrug resistancedue to mdr1 expression. Transfectants were selected with G418, andpossible changes in P-glycoprotein function were tested by determiningthe levels of resistance of individual transfectants to the cytotoxicdrugs vinblastine and colchicine.

All ten of the control transfectants obtained with pSV2neo had the samelevels of drug resistance as the recipient KB-8-5 cell line. Incontrast, twelve of fifteen transfectants obtained with construct 1 hadsignificantly decreased levels of drug resistance (in some cases lessthan one-half the resistance of KB-8-5). Five of eight transfectantsobtained with construct 2 also showed a significant decrease in drugresistance relative to control KB-8-5 cells. These results indicate thata short segment of P-glycoprotein, comprising only 4.5% of the proteinlength, can serve as a genetic suppressor element for P-glycoproteinfunction. There is no specific function presently associated with thissegment of P-glycoprotein, although this segment includes the amino acidresidue 185 known to be a determinant of the specificity ofP-glycoprotein-drug interactions.

These results demonstrate that short protein fragments without a knownfunction can serve as dominant negative inhibitors of the wild-typeprotein, suggesting that dominant negative inhibitors may be selectedfrom a library expressing random short fragments of the target protein.

EXAMPLE 2 Preparation of an Antiviral Genetic Suppressor Element Library

Lambda phage DNA was fragmented by partial digestion with DNaseI in thepresence of Mn⁺⁺ ions and NcoI linkers were added to the termini of theresulting fragments by blunt-end ligation after filling in the terminiwith T4 DNA polymerase and Klenow fragment of DNA polymerase I.Fragments of 350-450 bp size were then isolated after NcoI digestion andagarose gel electrophoresis. The fragment mixture was inserted into aplasmid expression vector pKK233-2, which carries a gene for ampicillinresistance and expresses inserted sequences using an IPTG-inducible trcpromoter and a specific translation initiation region. See Amann et al.,Gene 40: 183 (1985). The vector was modified to provide for appropriatetermination of translation of the inserted segment by insertion of theDNA sequence 5′ CATGGTGACTGACTGAAGCT 3′ into the NcoI and HindIII sitesof the polylinker. The ligated mixture was used to transform E. colistrain PLK-F′ (sensitive to lambda), and a library of approximately80,000 ampicillin-resistant clones was obtained.

EXAMPLE 3 Identification and Isolation of Genetic Suppressor Elements

To identify and isolate genetic suppressor elements in a libraryprepared as described in Example 2, the amplified library was tested forthe presence of clones resistant to infection by bacteriophage lambda. Alibrary comprising cells transformed with an insert-free pKK233-2 vectorwas used as a control. After IPTG induction, aliquots of 10⁶ cells fromthe amplified library and the control were infected with lambda phageand plated on ampicillin-containing plates. The multiplicity ofinfection was selected so as to allow for the survival of 1%-3% of theinfected control bacteria. After the first infection, there was no majordifference in the number of surviving cells between the library and thecontrol cells. Plasmid DNA was then extracted from the mixture ofapproximately 3×10⁴ library-derived colonies that survived phageinfection, and this DNA was used to transform plasmid-free bacteria. Thenew library was also infected with lambda, and this time approximately10% of the cells in the library were found to be resistant under theconditions of infection that allowed either 3% or 0.02% of the controlcells to survive. Plasmids were then isolated from 30 surviving coloniesand used individually to transform fresh E. coli cells. After infectionwith lambda, cells transformed with 28 of 30 selected plasmids showedresistance to lysis.

Parallel studies with the control plasmid showed no increase in thenumber of resistant colonies after three rounds of selection, indicatingthat the immunizing clones were specific to the lambda fragment library.Restriction enzyme analysis showed that almost all the plasmids carriedNcoI inserts of the expected size (350-450 bp). Based on the observedfrequency of the resistant cells, approximately 0.3% of the clones inthe original fragment library carried GSEs. Only a minority of thesuppressing and infected bacterial colonies showed chromosomalintegration of lambda sequences after infection, thus indicating thatinduction of lysogeny is not a major mechanism for protection by thesuppressing clones.

Another library was prepared as described in Example 2, except that theinsert fragments were of an average size of 600-700 bp. Although thislibrary also contained suppressing clones, their frequency was an orderof magnitude lower than in the 350-450 bp library.

These results demonstrate that random fragmentation of DNA homologous toa gene whose function is to be suppressed, followed by libraryconstruction and biological selection or screening, is a feasiblegeneral approach for the isolation of genetic suppressor elements.

EXAMPLE 4 Characterization of Genetic Suppressor Elements

Fifty-one of the isolated GSE clones were characterized by DNAsequencing. The sequenced GSEs fell into 11 classes, each classrepresenting a different region of the lambda genome. See FIG. 1. Thesuppression efficiency of different classes of GSE was evaluated by thefollowing tests. (a) Plating efficiency of transformed bacteria wasmeasured after lambda infection at high m.o.i. Bacteria transformed withany of the GSE showed either none or a minor (<2-fold) decrease in theplating efficiency. (b) The phage titer was determined by plaque assayusing the amounts of phage that produced 10⁹ plaques in controlbacteria. No plaques were discernible with most types of GSE, thoughsome GSE allowed for the formation of phage plaques at the incidence of10⁻⁵ to 10⁻⁷, apparently reflecting the appearance of GSE-insensitivemutant phage. (c) To determine the effect of GSEs on prophage induction,representative clones of each class were introduced into a lysogenicstrain of E. coli and the phage titer was determined after induction.Eight classes of GSE decreased the titer of the induced phage by threeor more orders of magnitude, but GSEs of the other three classes had noeffect on prophage induction.

Sense-oriented GSEs

Eight classes of GSE contained lambda gene fragments inserted in thesense orientation relative to the promoter. The inserted fragmentsencoded either partial or complete lambda proteins. Translation wasinitiated from the native initiation codon, from a linker-derivedinitiation codon that was in-frame with the coding sequence, or from aninitiation codon within the fragment. Two or more identical copies werefound for eight different GSEs. The most abundant class of GSE containedsequences of the gene Ea8.5, previously of unknown function. This classof GSE is described in Example 5.

Two sense-oriented classes of GSE, each represented by a single clone,contained truncated sequences from lambda genes having unknownfunctions. The first of these encoded the C-terminal 216 of 296 aminoacids encoded by the full-length Ea31 gene. The second GSE encoded theC-terminal 88 of 410 amino acids encoded by the full-length Ea47 gene.The coding sequence of each GSE was in frame with a translationinitiation codon from the linker. These GSEs inhibited infection oftransformed bacteria by lambda phage, but did not suppress lysogeninduction.

Another GSE class, represented by 2 clones, contained an intact cro genein sense orientation. Since cro encodes a regulatory protein thatsuppresses expression of lambda early genes, its GSE effect wasexpected.

Four classes of GSE encoded truncated forms of phage particle structuralproteins. One such GSE encoded the C-terminal 80 of 117 amino acidsencoded by the full-length FI gene, as well as the N-terminal 40 aminoof 117 amino acids encoded by the full-length FII gene. The FI and FIIgenes encode lambda head proteins. Another GSE-encoded the C-terminal159 of 198 amino acids encoded by the FII-length K gene, as well as theN-terminal 121 of 223 amino acids encoded by the full-length I gene. TheK and I genes encode lambda tail proteins.

Two other GSE classes encoded truncated forms of tail proteins V or G.The two clones of the first class encoded identical amino acid sequences(the first 145 of 256 amino acids of V protein), as did the two clonesof the second class (the first 113 of 140 amino acids of G protein). Inneither case, however, could the two clones be siblings, since theirnucleotide sequences were non-identical. To confirm the proteininterference mechanism of action, the V protein GSE was mutated tointroduce a nonsense mutation in the fourth codon. Introduction of thismutation abolished GSE activity.

Antisense-oriented GSEs

Three classes of GSE contained lambda gene sequences inserted inantisense orientation relative to the promoter. One such clone containedan internal segment of lambda gene A (positions 1050-1470), which isinvolved in DNA packaging. Two other classes of antisense GSEs wererepresented by multiple clones. The first class included 12non-identical clones encoding RNA complementary to the 5′ portion oflambda gene Q, which positively regulates lambda late transcription. AllGSEs in this class overlapped the naturally-occurring lambda antisensetranscript P_(aQ), which downregulates Q expression. None of these GSEsinitiated more than 70 bp upstream from the normal P_(aQ) promoter,although they contained downstream flanking sequences of variablelengths. Seven of these GSEs initiated within a 16 bp region.

Another class of antisense GSEs included four different GSEs thatencoded nearly identical antisense RNA sequences corresponding to the 3′end of the lambda gene CII, which regulates lysogeny, and the 5′ half oflambda gene O, which encodes a lambda replication protein. As shown inFIG. 2, each of these GSEs included the lambda origin of replication,located in the middle of lambda gene O, as well as thenaturally-occurring lambda antisense transcript oop, which iscomplementary to CII and normally suppresses CII. While these GSEssuppress lytic infection, overexpression of oop normally enhances lambdalytic infection. Two truncated variants of these GSEs were prepared todetermine whether some portion of the GSEs other than the oop sequenceswas responsible for the observed suppression. One variant lacked a 93 bpsegment encoding most of the oop sequence, but retained the 5′ portionof lambda gene O, including the lambda origin of replication. The othervariant lacked a 158 bp segment of lambda gene O, comprising the lambdaorigin of replication, but retained the oop sequence and the remainderof the 5′ of lambda gene O. Neither variant suppressed lambda infection,indicating that both the oop and gene O sequences, including the lambdaorigin of replication, were required for suppression.

Interpretation of Results

The GSEs characterized in these studies act by a variety of mechanisms.First, numerous GSEs encoded truncated versions of lambda structuralproteins, and thus apparently act as dominant negative mutants,interfering with phage particle assembly. Second, some GSEs encodeantisense RNAs that are complementary to required lambda genetranscripts. Since these GSEs contained naturally-occurring regulatoryantisense transcripts of lambda, this demonstrates that random fragmentselection of GSEs can be used to identify natural mechanisms of genesuppression. This is confirmed by a third type of GSE, which encodesintact regulatory proteins of lambda. Fourth, some GSEs encode antisenseRNAs that act by a suppression mechanism that is distinct from thetraditional antisense RNA mechanism of simple interference withstructural gene function. These GSEs encoding the oop/O gene antisenseRNAs likely interfere with DNA replication directly, since they coincidewith the lambda origin of replication. Such interference may result frominterference with RNA annealing that might be involved in initiation oflambda DNA replication.

Both sense-oriented and anti-sense oriented GSEs have shown coincidenceor near coincidence of termini among different clones, indicating strictsequence limitations for GSEs. This finding indicates that the randomfragment selection strategy provided by the invention is critical forsuccessfully obtaining GSEs. In addition, random fragment selection forGSEs that are larger or smaller than the 300-500 bp fragments used inthese studies can reveal additional classes of GSEs. Selection of veryshort GSEs that can be used to identify antisense oligonucleotide orpeptide sequences that can be synthesized chemically to producebioactive molecules is of particular interest.

EXAMPLE 5 Use of Random Fragment Selection of GSEs to Identify NovelGene Function

In the characterization studies described in Example 4, the mostabundant class of GSE contained sequences of the lambda gene Ea8.5inserted in sense orientation. The function of the Ea8.5 gene has beenpreviously unknown. It is transcribed in the delayed early stage oflytic infection, but is not required for either lytic or lysogenicinfection. The gene encodes a 93 amino acid protein. Some of the GSEsencoded intact Ea8.5 protein, while others encoded truncated proteins,missing 7 to 38 C-terminal or 3 to 10 N-terminal amino acids. Thesuppression effect was abolished by introduction of a frameshiftmutation into the second codon, indicating that Ea8.5 protein itself, inintact or truncated form, was required for suppression. Expression ofEa8.5 in a lysogenic strain did not suppress prophage induction,indicating that Ea8.5 acts at an initial stage of infection, such asphage entry into the host cell. Bacteria expressing Ea8.5 were deficientin maltose metabolism, as assayed on McConkey media with maltose, butwere proficient in galactose, lactose, mannose and arabinose metabolism.The malK-lamB RNA, from one of the three maltose operons of E. coli, wasabsent in bacteria expressing Ea8.5 protein, indicating that suppressionis associated with inhibition of the maltose operon encoding the lamBlambda receptor. GSEs encoding truncated Ea8.5 protein showed anincomplete but still significant suppression of malK-lamB RNA productionand maltose metabolism. We have also tested Ea8.5-transformed bacteriafor resistance to imm^(λ)h⁸⁰ a recombinant of phages lambda and φ80 thatenters the cell through a receptor different from LamB. Thetransformants were found to be sensitive to this phage, thus confirmingthe receptor-mediated mechanism of protection by Ea8.5 GSEs. Theseresults indicate that random fragment selection of GSEs can be used toidentify a previously unknown gene function.

EXAMPLE 6 Development of GSEs for Human Topoisomerase II

Topoisomerase II is a DNA unwinding enzyme that serves as a target formany anti-cancer drugs, including etoposide, doxorubicin and amsacrine.The enzyme normally acts by double-strand DNA cleavage, followed bystrand passage and religation of the breaks. Anti-cancer drugs causetrapping of the enzyme in complexes having double-strand breaks heldtogether by the enzyme, thereby leading to lethal damage in replicatingcells. Some cell lines that are resistant to anti-cancer drugs thatinteract with topoisomerase II have decreased expression of this enzyme.

Random fragment selection of GSEs requires transfer of the expressionlibrary into a very large number of recipient cells. Therefore, toprepare a random fragment library containing GSEs for topoisomerase II,the efficient retroviral vector system was chosen. Overlapping cDNAclones spanning the entire coding sequence for topoisomerase II weremixed and randomly fragmented into 250-350 bp fragments by DNase, asdescribed in Example 2. After ligation with a synthetic adaptorproviding translation initiation and termination codons, the fragmentmixture was amplified by PCR, using adaptor-derived primers. Theamplified mixture was cloned into the LNCX retroviral vector whichcontains a neo gene. Miller and Rosman, Biotechniqes 7:980-986 (1989). Afragment library containing 20,000 independent clones was obtained, andwas used to transfect amphotropic and ecotropic virus-packaging celllines derived from NIH 3T3 cells, to effect ping-pongreplication-mediated amplification of the virus. See Kozak and Kabat, J.Virol. 64: 3500-3508 (1990). This resulted in a random fragmentexpression library (RFEL), a set of recombinant retroviruses containinga representative mixture of inserts derived from topoisomerase II genesequences.

The uniformity of sequence representation in RFEL was monitored asfollows. NIH 3T3 cells were infected with virus-containing supernatant,followed 24 hours later by PCR amplification of integrated proviralinsert sequences in the presence of [³²P] alpha-dNTP. An aliquot of thePCR-amplified mixture was subjected to gel electrophoresis to establishthe absence of predominant bands. Another aliquot was used as a probefor a Southern blot of topoisomerase II cDNA digested with severalfrequently cutting restriction enzymes. A representative sequencemixture was obtained, as evidenced by the absence of a predominant bandin the first test, and uniform hybridization to all fragments in thesecond test.

RFEL was then used to infect HeLa cells, and the infectants wereselected with G418. Colonies of G418-resistant cells, having about 50-70cells each, were then exposed to etoposide at a concentration of 200ng/ml. Approximately 50 of 10,000 G418-resistant colonies were etoposideresistant, compared to a frequency of <10⁻⁴ when insertless retroviruseswere used as a control. Cell lines were isolated frometoposide-resistant colonies. Amphotropic and ecotropic packaging celllines producing RFEL were also selected for etoposide resistance. Virusfrom etoposide resistant packaging cell lines was used to infect HeLacells, which were then selected with G418. G418-resistant infectantswere challenged with three topoisomerase II-interactive anticancerdrugs: etoposide, teniposide and amsacrine. A high proportion ofinfected cells were resistant to all three drugs, thus demonstratingthat etoposide selection of mouse packaging cell lines has led to thegeneration of GSEs active in both human and mouse cells. Theseinfectants were also used to establish cell lines. RFEL-derived insertswere recovered from etoposide resistant cell lines by PCR and reclonedinto LNCX vector. The newly-derived clones were then individually testedfor the ability to confer resistance to etoposide upon transfection intoHeLa cells, to confirm the GSE activity of the corresponding inserts.

Sequence analysis of 26 different isolated clones revealed that 16 ofthem were inserted in antisense and 10 in sense orientation. Of the 10GSEs confirmed so far, 5 were sense and 5 antisense, as shown inTable 1. The sequences of the confirmed GSEs are shown in FIGS. 3A-3C.The sense-oriented inserts of the confirmed GSEs encode 37-99 amino acidlong topo II-derived peptides, initiating either from the ATG codonprovided by the adaptor, or from an internal ATG codon within the openreading frame of Topoisomerase II, located close to the 5′ end of theinsert in an appropriate context for translation initiation. Four of theconfirmed antisense GSEs come from the 3′ third of the cDNA and one fromthe 5′ end of cDNA, including the translation start site. Of theconfirmed sense-oriented GSEs, three are derived from the centralportion of the protein that includes the active site tyrosine-804 thatcovalently binds to DNA and the “leucine zipper” region involved indimerization of Topoisomerase II. One GSE peptide is derived from theregion near the N-terminus and another from the region near theC-terminus of the protein; no known functional sites are associated witheither segment.

These results establish that the principles for producing GSEs in aprokaryotic system (lambda phage in E. coli) can be extended to amammalian or human system through the use of an amphotropic retroviralvector system. As in the prokaryotic system, the GSEs obtained actaccording to multiple mechanisms. In addition, these results show thatGSEs produced from one mammalian species can be active in anothermammalian species. Finally, these results demonstrate that GSEs fortopoisomerase II are obtainable using a random fragment expressionlibrary. Such GSEs are useful for positive selection of geneticallymodified mammalian cells, in vitro, and for human gene therapy forrendering bone marrow resistant to anticancer drugs that interact withTopoisomerase II.

TABLE 1 CONFIRMED TOPOISOMERASE II-DERIVED GSE Orientation PositionPosition (Sense/ in of Clones Antisense) cDNA^(a) peptide^(b) 2VAntisense -18-145 Σ11 Sense 393-605 134-201 6 Sense 2352-2532 808-844 5Sense 2511-2734 846-911 Σ28 Sense 2603-2931 879-977 Σ2 Antisense3150-3343 Σ20 Antisense 3486-3692 39 Antisense 3935-4127 12S, Sense4102-4343 1368-1447 ΣVP Σ8 Antisense 4123-4342 ^(a)Position in the cDNAsequence of topoisomerase II; residues numbered as in Tsai-Pflugfelderet al., Proc. Natl. Acad. Sci. USA 85: 7177-7181 (1988). ^(b)Position ofthe peptide encoded by sense-oriented GSEs in the amino acid sequence oftopoisomerase II; translation assumed to initiate from the first ATGcodon in the correct open reading frame.

EXAMPLE 7 Preparation of GSEs that Abolish HLA Antigen Expression

Destruction of target cells by cytotoxic T lymphocytes requires thepresence of major histocompatibility (MHC, HLA) Class I antigens on thetarget cells for adhesion as well as for triggering of theantigen-specific T cell response. Masking of MHC Class I antigensprevents xenograft rejection of human donor cells in mouse recipients.Thus, target cells can be protected from immune destruction bydeliberate reduction of MHC Class I antigens on the surface of suchcells. Target cells resistant to destruction by cytotoxic T lymphocytesare useful for a variety of purposes. For example, they can be used ashuman tumor xenografts that can act as in vivo models for anticancerdrug testing in immunocompetent mice. Moreover, some such human tissueculture cells e.g., pancreatic cells can be used for tissuetransplantation into unmatched recipient patients.

Expression of MHC Class I antigen on the cell surface requiresco-expression of β₂-microglobulin, a highly conserved protein. Thus,both β₂-microglobulin and MHC Class I protein are targets forsuppression that leads to resistance to immune destruction. Mice thatare deficient in β₂-microglobulin production express little if any MHCClass I antigen on cell surfaces, yet are fertile and apparentlyhealthy, except for the absence of CD4⁻8⁺ T cells.

Tissue culture cells that are resistant to immune destruction areprepared by infection with a random fragment expression library for GSEsderived from β₂-microglobulin. The nucleotide sequence for humanβ₂-microglobulin was described by Gussow et al., J. Immunol. 139:3132-3138 (1987). The complete human β₂-microglobulin cDNA sequence isused to prepare RFEL, as described in Example 6, and infected cells areselected for G418 resistance. Infected cells are then selected forresistance to immune destruction by injection into immunocompetent mice.The selected cells are used to isolate the GSEs, as described in Example6. The isolated GSEs are then used to render other cell types resistantto immune destruction. Alternatively, the GSE library is prepared fromcDNA of all MHC Class I genes.

EXAMPLE 8 Preparation of a Normalized Random Fragment Library for TotalHuman cDNA

It is desirable to be able to obtain GSEs for any gene, the suppressionof which will have a desirable effect, without requiring specialknowledge of the gene structure or function. Examples of such genesinclude presently unknown tumor suppressor genes or genes thatpotentiate the cytotoxic action of anticancer drugs.

For isolation of GSEs corresponding to a mammalian gene that isexpressed at moderate or high levels, an RFEL of total cDNA can be used.However, for isolation of GSEs corresponding to genes that are expressedat low levels, the use of normalized cDNA libraries is desirable.Preparation of a normalized cDNA population has been described byPatanjali et al., Proc. Natl. Acad. Sci. USA 88:1943-1947 (1991).Poly(A)+ RNA is extracted from HeLa cells and randomly primed shortfragment cDNA is prepared. For purposes of preparing random fragmentlibraries the procedure is modified by ligating the cDNA to a syntheticadaptor providing translation initiation and termination codons,followed by PCR amplification, as described in Example 6. PCRs arecarried out in many separate reactions that are subsequently combined,in order to minimize random over- or underamplification of specificsequences and to increase the yield of the product. The PCR amplifiedmixture is then size-fractionated by gel electrophoresis and 300-500 bpfragments are taken.

The representation of different mRNA sequences is monitored by Southernblot hybridization of the mixture, using a series of six to eight probescorresponding to mRNAs of different abundance. Ribosomal DNA and β-actinare good high abundance probes, while c-myc and dhfr serve as moderateabundance probes and C-H-ras and c-K-ras are low abundance probes.Normalization is accomplished by denaturation and reannealing of thePCR-amplified cDNA, using 24, 48, 72, 96 and 120 hour time points forreannealing. Single and double stranded DNAs are then separated fromeach reannealed mixture by hydroxyapatite chromatography. Singlestranded DNA fractions from each time point are PCR-amplified usingadaptor derived primers and are analyzed by Southern hybridization forrelative abundance of different sequences. Selectiveunder-representation of the most abundant species may be avoided bymixing two library aliquots reannealed at different times at a ratiocalculated to give the most uniform representation.

The normalized cDNA population is then cloned into the LNCX retroviralvector, as described in Example 6. The library is then amplified byping-pong amplification, using a 1:1 mixture of ecotropic packaging cellline GP+E86 and amphotropic packaging cell line GP+envAm12, Markowitz etal., Virology 167: 400-406 (1988), in 10-15 separate batches to produceapproximately 106 independent clones per batch. We have obtained a yieldof amphotropic virus 11-12 days after infection of >10⁶ per 10 ml mediasupernatant from a single 100 mm plate. These amphotropic virus havefairly even representation of different fragments, but at later stagesindividual virus-producing clones begin to predominate, thereby makingsequence representation uneven. Uniform sequence representation ismonitored by rapid extraction of DNA from cells infected with packagingcell supernatant, followed by linker-specific PCR amplification andSouthern hybridization with different probes.

EXAMPLE 9 Use of Normalized Random Fragment GSE Libraries to IdentifyRecessive Genes

In order to obtain GSEs for any particular gene from a libraryrepresenting total mRNA, it is necessary to be able to generate a verylarge library. Somatic tissues of higher eukaryotes express mRNA forabout 10,000 genes. For an average mRNA length of about 2.5 kb, thetotal mRNA or cDNA complexity for a given tissue type is about 25,000kb. We have discovered that in a library prepared from a 6 kb cDNAencoding human topoisomerase II, approximately 1 in 200 clones carriedGSEs. This corresponds to a frequency of about one GSE for every 33clones for every kilobase of library complexity. Thus, for a library of25,000 kb complexity, the frequency of GSEs for a particular gene isabout 1 in 825,000 clones, or approximately 10⁻⁶.

To be certain that at least one GSE is present for every gene, a libraryof about 10⁷ independent clones is prepared, as described in Example 7.Some twenty 150 mm plates, each having about 50,000 colonies, issufficient for screening of about 10⁶ infected HeLa cells. Thus, 10-15batches of such twenty plate selections are sufficient for isolation ofa GSE for any desired recessive gene for which a negative selection ispossible (e.g., 200 ng/ml etoposide for topoisomerase II GSES). As inExample 6, G418 selection is followed by the negative selection oncolonies having 50-70 cells. Depending on the background level ofresistance to the negative selection, resistant colonies are processedindividually or mixed and subjected to another round of recloning andGSE selection. Inserts of GSEs are then used to identify the gene oforigin by sequencing and data base comparison, by use as a probe inconventional cDNA cloning, or by use in cDNA cloning by the “anchoredPCR” procedure. See Ohara et al., Proc. Natl. Acad. Sci. USA 86:5673-5677 (1989).

EXAMPLE 10 Derivation of Anti-HIV-1 Genetic Suppressor Elements

Cloned human immunodeficiency virus-1 (HIV-1) cDNA is digested withDNase I, filled-in, fitted with linkers and size-selected, as describedin Example 2. The fragment mixture is transferred into a retroviralexpression vector that carries a dominant selectable marker and iscapable of infecting human T cells. The HIV fragment/retroviral vectorlibrary is used to infect a human T cell line that is susceptible tokilling by HIV-1 and infected cells are selected for the presence of thedominant marker. The mixture of selected cells is exposed to HIV-1, andcytopathic effect is allowed to develop to completion. Surviving cellsare expanded and their DNA is isolated. DNA sequences corresponding toHIV-1 fragments are obtained by amplification of isolated cellular DNAusing the polymerase chain reaction (PCR) with primers specific for theretroviral vector on either side of the insert.

PCR-generated DNA fragments are fitted with linkers and transferred tothe same retroviral vector that was used to prepare the first library tocreate a secondary library. The same T cell line that was used for theinitial library is then infected with the secondary library. Infectedcells are selected for the presence of the dominant marker andindividual selected clones are tested for resistance to killing byHIV-1. Resistant clones, containing putative anti-HIV-1 GSEs are usedfor the isolation of the putative GSE by the polymerase chain reaction,as described above. The candidate GSEs are then individually insertedinto the same retroviral vector and tested for the ability to protectT-cells against cytopathic effects of HIV-1.

EXAMPLE 11 Derivation of Anti-Tobacco Mosaic Virus (TMV) GeneticSuppressor Elements

Total TMV cDNA is randomly fragmented as described in Example 2. Thefragment mixture is then transferred into an expression vectorcontaining a neomycin phosphotransferase II gene such that the invertedfragment is transcribed, initiating from the cauliflower mosaic virus35S promoter and terminating in the polyadenylation signal from thenopaline synthase gene. Leaf disks of tobacco are inoculated withAgrobacterium tumefaciens cells containing the expression library.Transformed cells are selected in culture for kanamycin resistance.Kanamycin resistant cells are then exposed in culture to TMV andcytopathic effect is allowed to develop. DNA is collected fromtransformed TMV-resistant cells and the insert fragments are amplifiedby the polymerase chain reaction, using primers homologous to the DNAsequences adjacent to the insert site. Amplified sequences aretransferred into the same expression vector as used to make the initiallibrary and again used to transform A. tumefaciens. Tobacco leaf disksare once again inoculated with the library in A. tumefaciens andkanamycin-resistant cells are again tested for TMV resistance.Individual TMV-resistant clones are used for the isolation of GSEs bythe polymerase chain reaction, as described above. Candidate GSEs arethen used to prepare individual GSE expression vectors, which areinserted in A. tumefaciens to inoculate tobacco leaf disks. Inoculatedleaf disks are selected for kanamycin resistant cells, from whichself-pollinated individual seedlings are produced and tested for TMVresistance.

11 164 base pairs nucleic acid single linear cDNA NO YES not provided 1GTGTCTGGGC GGAGCAAAAT ATGTTCCAAT TGTGTTTTCT TTTGATAGAT TCTTTCAACA 60GACAGTCTTT TCTTAGCATC TTCATTTTTC TTTATTTTGT TGACTTGCAT ATTTTCATTT 120ACAGGCTGCA ATGGTGACAC TTCCATGGTG ACGGTCGTGA AGGG 164 213 base pairsnucleic acid single linear cDNA NO YES not provided 2 TGAAAAGATGTATGTCCCAG CTCTCATATT TGGACAGCTC CTAACTTCTA GTAACTATGA 60 TGATGATGAAAAGAAAGTGA CAGGTGGTCG AAATGGCTAT GGAGCCAAAT TGTGTAACAT 120 ATTCAGTACCAAATTTACTG TGGAAACAGC CAGTAGAGAA TACAAGAAAA TGTTCAAACA 180 GACATGGATGGATAATATGG GAAGAGCTGG TGA 213 181 base pairs nucleic acid single linearcDNA NO YES not provided 3 GCCCATTGGT CAGTTTGGTA CCAGGCTACA TGGTGGCAAGGATTCTGCTA GTCCACGATA 60 CATCTTTACA ATGCTCAGCT CTTTGGCTCG ATTGTTATTTCCACCAAAAC ATGATCACAC 120 GTTGAAGTTT TTATATGATG ACAACCAGCG TGTTGAGCCTGAATGGTACA TTCCTATTAT 180 T 181 224 base pairs nucleic acid singlelinear cDNA NO YES not provided 4 TGAATGGTAC ATTCCTATTA TTCCCATGGTGCTGATAAAT GGTGCTGAAG GAATCGGTAC 60 TGGGTGGTCC TGCAAAATCC CCAACTTTGATGTGCGTGAA ATTGTAAATA ACATCAGGCG 120 TTTGATGGAT GGAGAAGAAC CTTTGCCAATGCTTCCAAGT TACAAGAACT TCAAGGGTAC 180 TATTGAAGAA CTGGCTCCAA ATCAATATGTGATTAGTGGT GAAG 224 329 base pairs nucleic acid single linear cDNA NOYES not provided 5 TGCGTGAAAT TGTAAATAAC ATCAGGCGTT TGATGGATGGAGAAGAACCT TTGCCAATGC 60 TTCCAAGTTA CAAGAACTTC AAGGGTACTA TTGAAGAACTGGCTCCAAAT CAATATGTGA 120 TTAGTGGTGA AGTAGCTATT CTTAATTCTA CAACCATTGAAATCTCAGAG CTTCCCGTCA 180 GAACATGGAC CCAGACATAC AAAGAACAAG TTCTAGAACCCATGTTGAAT GGCACCGAGA 240 AGACACCTCC TCTCATAACA GACTATAGGG AATACCATACAGATACCACT GTGAAATTTG 300 TTGTGAAGAT GACTGAAGAA AAACTGGCA 329 194 basepairs nucleic acid single linear cDNA NO YES not provided 6 CACTCTTTTCAGTTTCCTTT TCGTTGTCAC TCTCTTCATT TTCTTCTTCA TCTGGAACCT 60 TTTGCTGGGCTTCTTTCCAG GCCTTCACAG GATCCGAATC ATATCCCCTC TGAATCAGAA 120 CTTTAATTAATTCTTTCTTA GGCTTATTTT CAATGATTAT TTTGCCATCT ATTTTCTCAT 180 AGATAAAGCGAGCC 194 206 base pairs nucleic acid single linear cDNA NO YES notprovided 7 TCTGCCTCTG CTTTCATTTC TATGGTTATT CGTGGAATGA CTCTTTGACCACGCGGAGAA 60 GGCAAAACTT CAGCCATTTG TGTTTTTTTC CCCTTGGCCT TCCCCCCTTTCCCAGGAAGT 120 CCGACTTGTT CATCTTGTTT TTCCTTGGCT TCAACAGCCT CCAATTCTTCAATAAATGTA 180 GCCAAGTCTT CTTTCCACAA ATCTGA 206 194 base pairs nucleicacid single linear cDNA NO YES not provided 8 GACACGACAC TTTTCTGTGGTTTCAGTTCT TTGTTACTAA GTTTTGGGGA AGTTTTGGTC 60 TTAGGTGGAC TAGCATCTGATGGGACAAAA TCTTCATCAT CAGTTTTTTC ATCAAAATCT 120 GAGAAATCTT CATCTGAATCCAAATCCATT GTGAATTTTG TTTTTGTTGC TGCTCTCCGT 180 GGCTCTGTTT CTCG 194 242base pairs nucleic acid single linear cDNA NO YES not provided 9CTGAAACCAC AGAAAAGTGT CGTGTCAGAC CTTGAAGCTG ATGATGTTAA GGGCAGTGTA 60CCACTGTCTT CAAGCCCTCC TGCTACACAT TTCCCAGATG AAACTGAAAT TACAAACCCA 120GTTCCTAAAA AGAATGTGAC AGTGAAGAAG ACAGCAGCAA AAAGTCAGTC TTCCACCTCC 180ACTACCGGTG CCAAAAAAAG GGCTGCCCCA AAAGGAACTA AAAGGGATCC AGCTTTGAAT 240 TC242 220 base pairs nucleic acid single linear cDNA NO YES not provided10 AATTCAAAGC TGGATCCCTT TTAGTTCCTT TTGGGGCAGC CCTTTTTTTG GCACCGGTAG 60TGGAGGTGGA AGACTGACTT TTTGCTGCTG TCTTCTTCAC TGTCACATTC TTTTTAGGAA 120CTGGGTTTGT AATTTCAGTT TCATCTGGGA AATGTGTAGC AGGAGGGCTT GAAGACAGTG 180GTACACTGCC CTTAACATCA TCAGCTTCAA GGTCTGACAC 220 20 base pairs nucleicacid single linear cDNA NO not provided 11 CATGGTGACT GACTGAAGCT 20

We claim:
 1. A method for producing GSEs corresponding to recessivegenes that, when inactivated by GSEs, confer a selectable or screenablephenotype upon a cell having such inactive genes, the method comprisingthe steps of: (a) obtaining genomic DNA or a total cDNA population fromthe cells; (b) randomly fragmenting the genomic DNA or total cDNApopulation to produce random DNA fragments; (c) ligating the random DNAfragments to synthetic adaptors to produce amplifiable random DNAfragments; (d) cloning the amplified mixture of random DNA fragmentsinto a suitable expression vector having a selectable marker to producea random fragment expression library; (e) transferring the randomfragment expression library into appropriate the random fragmentexpression library into appropriate target cells; (f) selecting thetarget cells for the presence of the selectable marker present in theexpression vector to obtain target cells having the selectable marker;(g) selecting or screening the target cells having the selectable markerfor the selectable or screenable phenotype conferred upon the cells byinactivation of a recessive gene by a GSE; (h) recovering the GSE fromthe target cell having the selectable or screenable phenotype.
 2. Amethod according to claim 1, wherein the target cells are mammaliancells.
 3. A method according to claim 1, wherein the target cells arebacterial cells.
 4. A method according to claim 1, wherein the targetcells are plant cells.
 5. A method according to claim 1, wherein thetotal cDNA population is a normalized cDNA population.
 6. A methodaccording to claim 1, wherein the GSE is a sense-oriented GSE encoding apeptide.
 7. A method according to claim 1, wherein the GSE is anantisense-oriented GSE encoding an antisense RNA.
 8. A method ofobtaining genetic suppressor elements (GSEs) comprising the steps of:(a) randomly fragmenting DNA homologous to a gene to be suppressed, toyield DNA fragments; (b) transferring the DNA fragments to an expressionvector to yield a library, wherein the expression vector is capable ofexpressing the DNA fragments in a living cell in which gene suppressioncan be selected or screened; (c) genetically modifying living cells byintroducing the genetic suppressor elements library into the livingcells; (d) isolating or enriching for genetically modified living cellscontaining genetic suppressor elements by selecting or screening forgene suppression, and; (e) obtaining the genetic suppressor element fromthe genetically modified cells.